Reasons for mis-expression - (Aug/12/2011 )
I am writing my bachelor thesis and I need some suggestions and support.
The important context: I did experiments where I synthesized cDNA out of Arabidopsis mRNA. Then I cloned it into Entry Clones to insert the sequence via LR-Reaction (Gateway Cloning) into a destination vector directly behind a fluorophor. So the expressed protein would have a fluorophor at its C-terminus. I did this with 10 cDNA sequences, each coding for a different protein. Each coding sequence was inserted into 3 different destination vectors with different fluorophors. Then I transformed them into bacteria and induced the gene expression in Tobacco plants. 9 proteins with 3 different fluorophors (=27) worked well. 1 Protein didn't work with any of the 3 different fluorophors (no fluorescence was detectable. What could be the reason?
My suggestions would be
1) Frameshift after the LR-Reaction in the recombination sites(unlikely that it happened for all 3 different destination vectors)
2) Mutation in the coding sequence (I sequenced the entry clones and there was no mutation)
3) Experimental error (I co-expressed one of the other fluorescence-tagged proteins and it worked)
4) Heterologous tobacco plant was not able to fold the protein correctly
Do my suggestions make sense?
I hope you can provide me with some ideas!
Is it possible that some regulatory sequences are missing because I used the cDNA instead of genomic DNA?
I forgot to mention that the genes were expressed under a inducible non-endogenous promotor.